Patent Application
20250011735
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 25, 2022, is named CBTH-11-PCT_SL.txt and is 215,720 bytes in size.
The present application generally relates to recombinant enzymes and genes encoding those enzymes. More specifically, the application provides recombinant geranyl pyrophosphate synthase genes and enzymes that function in yeast.
Cannabinoids are a class of organic small molecules of meroterpenoid structures found in the plant genus Cannabis. The small molecules are currently under investigation as therapeutic agents for a wide variety of health issues, including epilepsy, pain, and other neurological problems, and mental health conditions such as depression, PTSD, opioid addiction, and alcoholism.
While it is known that cannabinoids may be obtained via biosynthesis in plant species, there are many problems associated with the synthesis of such molecules which need to be overcome, including problems with large-scale manufacturing, purification, and heterologous expression for biosynthesis.
Terpenes and related terpenoids are another class of organic small molecules of commercial value. Terpenes may be used for flavors, fragrances, and are the major component of essential oils. Like cannabinoids, they are mostly produced in plants and are subject to the same difficulties as cannabinoids when produced in large quantities. Similarly, other plant derived terpenes may be produced from the same precursor molecules. These include alkaloids like salvinorin, carotenoids and mono, sequi and diterpenoids.
Producing terpenoids, including cannabinoids, in recombinant yeast is a promising solution to the above problems. See, e.g., U.S. patent applications Ser. No. 16/553,103, Ser. No. 16/553,120, Ser. No. 16/558,973, Ser. No. 17/068,636 and 63/053,539; U.S. Pat. No. 10,435,727; and US Patent Publications 2020/0063170 and 2020/0063171, all incorporated by reference.
Provided is a nucleic acid comprising a recombinant bacterial or archaeal geranyl pyrophosphate synthase (GPPS) gene, codon optimized for production in yeast.
Also provided is a yeast cell comprising an expression cassette comprising the above nucleic acid. In these embodiments, the yeast cell is capable of expressing a recombinant GPP synthase encoded by the above nucleic acid.
Additionally provided is a method of producing a terpene or a cannabinoid in a yeast, the method comprising incubating the above yeast cell in a manner sufficient to produce the terpene or cannabinoid.
To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:
Conservative amino acid substitutions: As used herein, when referring to mutations in a protein, “conservative amino acid substitutions” are those in which at least one amino acid of the polypeptide encoded by the nucleic acid sequence is substituted with another amino acid having similar characteristics. Examples of conservative amino acid substitutions are ser for ala, thr, or cys; lys for arg; gln for asn, his, or lys; his for asn; glu for asp or lys; asn for his or gln; asp for glu;
pro for gly; leu for ile, phe, met, or val; val for ile or leu; ile for leu, met, or val; arg for lys; met for phe; tyr for phe or trp; thr for ser; trp for tyr; and phe for tyr.
Functional variant: The term “functional variant,” as used herein, refers to a recombinant enzyme such as a GPPS that comprises a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequences of the parent protein and that is still capable of performing an enzymatic function (e.g., synthesis of GPP) of the parent enzyme. In other words, the modifications in the amino acid and/or nucleotide sequence of the parent enzyme may cause desirable changes in reaction parameters without altering fundamental enzymatic function encoded by the nucleotide sequence or containing the amino acid sequence. The functional variant may have conservative change including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may comprise natural as well as non-natural nucleotides and amino acids. Also envisioned is the use of amino acid analogs, e.g. amino acids not DNA or RNA encoded in biological systems, and labels such as fluorescent dyes, radioactive elements, electron dense agents, or any other protein modification, now known or later discovered.
Recombinant nucleic acid and recombinant protein: As used herein, a recombinant nucleic acid or protein is a nucleic acid or protein produced by recombinant DNA technology, e.g., as described in Green and Sambrook (2012).
Polypeptide, protein, and peptide: The terms “polypeptide,” “protein,” and “peptide” are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of greater than two amino acids. Unless otherwise specified, the terms “polypeptide,” “protein,” and “peptide” also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, and the like. Modifications also include intra-molecular crosslinking and covalent attachment of various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, and the like. In addition, modifications may also include protein cyclization, branching of the amino acid chain, and cross-linking of the protein. Further, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide.
The term “protein” or “polypeptide” may also encompass a “purified” polypeptide that is substantially separated from other polypeptides in a cell or organism in which the polypeptide naturally occurs (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% frec of contaminants).
Primer, probe and oligonucleotide: The terms “primer,” “probe,” and “oligonucleotide” may be used herein interchangeably to refer to a relatively short nucleic acid fragment or sequence. They can be DNA, RNA, or a hybrid thereof, or chemically modified analogs or derivatives thereof. Typically, they are single-stranded. However, they can also be double-stranded having two complementing strands that can be separated apart by denaturation. In certain aspects, they are of a length of from about 8 nucleotides to about 200 nucleotides. In other aspects, they are from about 12 nucleotides to about 100 nucleotides. In additional aspects, they are about 18 to about 50 nucleotides. They can be labeled with detectable markers or modified in any conventional manners for various molecular biological applications.
Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Various vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.”
Linker: The term “linker” refers to a short amino acid sequence that separates multiple domains of a polypeptide. In some embodiments, the linker prohibits energetically or structurally unfavorable interactions between the discrete domains.
Cannabinoid: As used herein, the term “cannabinoid” refers to a family of structurally related meroterpenoid molecules, all products of a common biosynthesis pathway.
Terpenoid: As used herein, the term “terpenoid” refers to a family of structurally related organic molecules derived from the 5-carbon compound isoprene, and the isoprene polymers called terpenes.
Codon optimized: As used herein, a recombinant gene is “codon optimized” when its nucleotide sequence is modified to accommodate codon bias of the host organism to improve gene expression and increase translational efficiency of the gene.
Expression cassette: As used herein, an “expression cassette” is a nucleic acid that comprises a gene and a regulatory sequence operatively coupled to the gene such that the promoter drives the expression of the gene in a cell. An example is a gene for an enzyme with a promoter functional in yeast, where the promoter is situated such that the promoter drives the expression of the enzyme in a yeast cell.
An important precursor molecule in the biosynthesis of cannabinoids and terpenes is geranyl pyrophosphate (GPP), also called geranyl diphosphate (
For a diterpenoid product such as the alkaloid salvinorin. GPP is modified by enzymes of the salvinorin biosynthesis pathway to create first, clerodienyl diphosphate or kolavenol diphosphate, as depicted in
For biosynthesis of the GPP derived terpene thujone, GPP is first converted to sabinene by sabinene synthase (Kshatriya, 2020). See
Diterpenoids such as carotenoids are derived from GGPP. First, GGPP is converted to phytoene by phytoene synthase, then phytoene to lycopene, beta carotene, canthaxanthin, astaxanthin and derivatives of these molecules (
It would therefore be useful to utilize GPP synthase (GPPS) in recombinant systems such as yeast to produce cannabinoids and other terpenoid compounds.
Thus, provided is a nucleic acid comprising a recombinant bacterial or archacal geranyl pyrophosphate synthase (GPPS) gene, codon optimized for production in yeast. Nonlimiting examples of such nucleic acids include GPPS genes having SEQ ID NOs: 1-46, encoding proteins having amino acid SEQ ID NOs: 47-92, respectively (Table 1). These bacterial GPP synthase (bkGPPS) enzymes and archacal GPP synthase (rkGPPS) enzymes have the capacity to synthesize GPP, NPP, FPP and/or GGPP in a recombinant host. Because they are codon optimized, they catalyze the production of GPP, NPP, FPP and/or GGPP more efficiently and with higher yield than the naturally occurring enzymes from which they are derived. The codon optimization is specific for a particular host. Additional enzymes may be selected from bacterial and archaeal hosts from a wide variety of habitats in order to match the conditions under which they will be utilized industrially to maximize or maintain enzymatic activity. For example, if the fermentation is to be run at high temperature, it may be beneficial to select a sequence derived from a thermophilic bacterium or archaeon.
The nucleic acid sequences in Table 1 having SEQ ID NOs: 1-46 are codon optimized to improve expression using techniques as disclosed in U.S. Pat. No. 10,435,727, which is incorporated herein by reference in its entirety. SEQ ID NOs: 1-24 are derived from bacterial GPPS (“bkGPP”) and SEQ ID NOs: 25-46 are derived from archaeal GPPS (“rkGPP”).
More specifically, optimized nucleotide sequences are generated based on a number of considerations: (1) For each amino acid of the recombinant polypeptide to be expressed, a codon (triplet of nucleotide bases) is selected based on the frequency of each codon in the Saccharomyces cerevisiae genome; the codon can be chosen to be the most frequent codon or can be selected probabilistically based on the frequencies of all possible codons. (2) In order to prevent DNA cleavage due to a restriction enzyme, certain restriction sites are removed by changing codons that cover those sites. (3) To prevent low-complexity regions, long repeats (sequences of any single base longer than five bases) are modified. (2) and (3) are performed recursively to ensure that codon modification does not lead to additional undesirable sequences. (4) A ribosome binding site is added to the N-terminus. (5) A stop codon is added.
Biosynthesis of sesquiterpenes utilize farnesyl pyrophosphate (
Additionally, the class of terpenes known as diterpenes is derived from geranylgeranyl pyrophosphate (
In some embodiments, the nucleic acid comprises a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the thirty-five sequences of SEQ ID NOs: 1-46, or its complement, or an RNA equivalent thereof.
In other embodiments, the nucleic acids provided herein encode an enzymatically active GPPS comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or conservative amino acid substitution to any one of the forty-six sequences of SEQ ID NOs: 47-92. These polypeptides are capable of synthesizing GPP, FPP, and/or GGPP.
In some embodiments, the GPPS gene is derived from a bacterium. It is envisioned that a GPPS from any bacterium now known or later discovered can be utilized in the present invention. For example, the bacterium can be from phylum Abditibacteriota, including class Abditibacteria, including order Abditibacteriales; phylum Abyssubacteria or Acidobacteria, including class Acidobacteriia, Blastocatellia, Holophagac, Thermoanacrobaculia, or Vicinamibacteria, including order Acidobacteriales, Bryobacterales, Blastocatellales, Acanthopleuribacterales, Holophagales, Thermotomaculales, Thermoanacrobaculales, or Vicinamibacteraceae; phylum Actinobacteria, including class Acidimicrobiia, Actinobacteria, Actinomarinidac, Coriobacteriia, Nitriliruptoria, Rubrobacteria, or Thermoleophilia, including orders Acidimicrobiales, Acidothermales, Actinomycetales, Actinopolysporales, Bifidobacteriales, Nanopelagicales, Catenulisporales, Corunebacteriales, Cryptosporangiales, Frankiales, Geodermatophilales, Glycomycetales, Jiangellales, Micrococcales, Micromonosporales, Nakamurellales, Propionibacteriales, Pseudonocardiales, Sporichthyales, Streptomycetales, Streptosporangiales, Actinomarinales, Coriobacteriales, Eggerthellales, Egibacterales, Egicoccales, Euzebyales, Nitriliruptorales, Gaiellales, Rubrobacterales, Solirubrobacterales, or Thermoleophilales; phylum Aquificac, including class Aquificae, including order Aquificales or Desulfurobacteriales; phylum Armatimonadetes, including class Armatimonadia, including order Armatimonadales, Capsulimonadales, Chthonomonadetes, Chthonomonadales, Fimbriimonadia, or Fimbriimonadales; phylum Aureabacteria or Bacteroidetes, including class Armatimonadia, Bacteroidia, Chitinophagia, Cytophagia, Flavobacteria, Saprospiria or Sphingobacteriia, including order Bacteroidales, Marinilabiliales, Chitinophagales, Cytophagales, Flavobacteriales, Saprospirales, or Sphingopacteriales; phylum Balneolacota, Caldiserica, Calditrichacota, or Chlamydiae, including class Balneolia, Caldisericia, Calditrichac, or Chlamydia, including order Balncolales, Caldisericales, Calditrichales, Anoxychlamydiales, Chlamydiales, or Parachlamydiales; phylum Chlorobi or Chloroflexi, including class Chlorobia, Anacrolineac, Ardenticatenia, Caldilineae, Thermofonsia, Chloroflexia, Dehalococcoidia, Ktedonobacteria, Tepidiformia, Thermoflexia, Thermomicrobia, or Sphaerobacteridae, including order Chlorobiales, Anacrolineales, Ardenticatenales, Caldilineales, Chloroflexales, Herpetosiphonales, Kallotenuales, Dehalococcoidales, Dehalogenimonas, Ktedonobacterales, Thermogemmatisporales, Tepidiformales, Thermoflexales, Thermomicrobiales, or Sphaerobacterales; phylum Chrysiogenetes, Cloacimonetes, Coprothermobacterota, Cryosericota, or Cyanobacteria, including class Chrysiogenetes, Coprothermobacteria, Glocobacteria, or Oscillatoriophycideac, including order Chrysiogenales, Coprothermobacterales, Chroococcidiopsidales, Glocoemargaritales, Nostocales, Pleurocapsales, Spirulinales, Synechococcales, Glocobacterales, Chroococcales, or Oscillatoriales; phyla: Eferribacteres, Deinococcus-thermus, Dictyoglomi, Dormibacteracota, Elusimicrobia, Eremiobacteracota, Fermentibacteria, or Fibrobacteres, including class Deferribacteres, Deinococci, Dictyoglomia, Elusimicrobia, Endomicrobia, Chitinispirillia, Chitinivibrionia, or Fibrobacteria, including order Deferribacterales, Deinococcales, Thermales, Dictyoglomales, Elusimicrobiales, Endomicrobiales, Chitinspirillales, Chitinvibrionales, Fibrobacterales, or Fibromonadales; phylum Firmicutes, Fusobacteria, Gemmatimonadetes, or Hydrogenedentes, including class Bacilli, Clostridia, Erysipelotrichia, Limnochordia, Negativicutes, Thermolithobacteria, Tissierellia, Fusobacteriia, Gemmatimonadetes, Longimicrobia, including order Bacillales, Lactobacillus, Borkfalkiales, Clostridiales, Halanacrobiales, Natranacrobiales, Thermoanaerobacterales, Erysipelotrichales, Limnochordales, Acidaminococcales, Selenomonadales, Veillonellales, Thermolithobacterales, Tissierellales, Fusobacteriales, Gemmatimonadales, or Longimicrobia; phylum Hydrogenedentes, Ignavibacteriac, Kapabacteria, Kiritimatiellacota, Krumholzibacteriota, Kryptonia, Latescibacteria, LCP-89, Lentisphacrae, Margulisbacteria, Marinimicrobia, Melainabacteria, Nitrospinac, or Omnitrophica, including class Ignavibacteria, Kiritimatiellac, Krumholzibacteria, Lentisphacria, Oligosphacria, or Nitrospinae, including order Ignavibacteriales, Kiritimatiellales, Krumholzibacteriales, Lentisphaerales, Victivallales, Oligosphaerales, or Nitrospinia; phylum Omnitrophica or Planctomycetes, including class Brocadiac, Phycisphaerae, Planctomycetia, or Phycisphacrales, including order Sedimentisphaerales, Tepidisphaerales, Gemmatales, Isosphaerales, Pirellulales, or Planctomycetales; phylum Proteobacteria including class Acidithiobacillia, Alphaproteobacteria, Betaproteobacteria, Lambdaproteobacteria, Muproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Gammaproteobacteria, Hydrogenophilalia. Oligoflexia, or Zetaproteobacteria, including order Acidithiobacillales, Caulobacterales, Emcibacterales, Holosporales, Iodidimonadales, Kiloniellales, Kopriimonadales, Kordiimonadales, Magnetococcales, Micropepsales, Minwuiales, Parvularculales, Pelagibacterales, Rhizobiales, Rhodobacterales, Rhodospirillales. Rhodothalassiales, Rickettsiales, Sneathiellales, Sphingomonadales, Burkholderiales, Ferritrophicales, Ferrovales, Neisseriales, Nitrosomonadales, Procabacteriales, Rhodocyclales, Bradymonadales, Acidulodesulfobacterales, Desulfarculales, Desulfobacterales, Desulfovibrionales, Desulfurellales, Desulfuromonadales, Myxococcales, Syntrophobacterales, Campylobacterales, Nautiliales, Acidiferrobacterales, Acromonadales, Alteromonadales, Arenicellales, Cardiobacteriales, Cellvibrionales, Chromatiales, Enterobacterales, Immundisolibacterales, Legionellales, Methylococcales, Nevskiales, Oceanospirillales, Orbales, Pasteurellales Pseudomonadales, Salinisphaerales, Thiotrichales, Vibrionales, Xanthomonadales, Hydrogenophilales, Bacteriovoracales, Bdellovibrionales, Oligoflexales, Silvanigrellales, or Mariprofundales; phylum Rhodothermacota, Saganbacteria, Sericytochromatia, Spirochactes, Synergistetes, Tectomicrobia, or Tencricutes, including class Rhodothermia, Spirochactia, Synergistia, Izimaplasma, or Mollicutes, including order Rhodothermales, Brachyspirales, Brevinematales, Leptospirales, Spirochactales, Synergistales, Acholeplasmatales, Anacroplasmatales, Entomoplasmatales, or Mycoplasmatales; phylum Thermodesulfobacteria, Thermotogac, Verrucomicrobia, or Zixibacteria, including class Thermodesulfobacteria, Thermotogac, Methylacidiphilae, Opitutac, Spartobacteria, or Verrucomicrobiac, including order Thermodesulfobacteriales, Kosmotogales, Mesoaciditogales, Petrotogales, Thermotogales, Methylacidiphilales, Opitutales, Puniceicoccales, Xiphinematobacter, Chthoniobacterales, Terrimicrobium, or Verrucomicrobiales.
In other embodiments, the GPPS gene is derived from an archaeon. It is envisioned that a GPPS from any archaeon now known or later discovered can be utilized in the present invention. For example, the bacterium can be from phylum Euryarchaeota, including class Archaeoglobi, Hadesarchaea, Halobacteria, Methanobacteria, Methanococci, Methanofastidiosa, Methanomicrobia, Methanopyri, Nanohaloarchaea, Theionarchaea, Thermococci, or Thermoplasmata, including order Archaeoglobales, Hadesarchaeales, Halobacteriales, Methanobacteriales, Methanococcales, Methanocellales, Methanomicrobiales, Methanophagales, Methanosarcinales, Methanopyrales, Thermococcales, Methanomassiliicoccales, Thermoplasmatales, or Nanoarchaeales; DPANN superphylum, including subphyla Aenigmarcheota, Altiarchaeota, Diapherotrites, Micrarchaeota, Nanoarchaeota, Pacearchaeota, Parvarchaeota, or Woesearchaeota; TACK superphylum, including subphylum Korarchaeota, Crenarchaeota, Aigarchaeota, Geoarchaeota, Thaumarchaeota, or Bathyarchaeota; Asgard superphylum including subphylium Odinarchaeota, Thorarchaeota, Lokiarchaeota, Helarchaeota, or Heimdallarchaeota.
The nucleic acids of the present invention can further comprise additional nucleotide sequences or other molecules. In some embodiments, the additional sequences encode additional amino acids present when the nucleic acid is translated, encoding, for example, an additional protein domain, with or without a linker sequence, creating a fusion protein. Other examples are localization sequences, i.e., signals directing the localization of the folded protein to a specific subcellular compartment or membrane.
In some embodiments, any of the codon optimized nucleic acids having sequences SEQ ID NOs: 1-46 are have, at the 5′ end, a nucleic acid encoding codon optimized cofolding peptides to create a fusion protein, e.g., having SEQ ID NOs: 93-97 (Table 2), joining the sequences together to form a fusion polypeptide, e.g., having the amino acid sequence of SEQ ID NO:98-102 fused at the N terminus of any of the polypeptides having SEQ ID NO:47-92, generating recombinant fusion polypeptides.
Other additional amino acids that can be added to the GPPS of the present invention include various yeast protein tags and modifiers. See e.g. http://parts.igem.org/Yeast.
In other embodiments, the nucleic acid comprises additional nucleotide sequences that are not translated. Examples include promoters, terminators, barcodes, Kozak sequences, targeting sequences, and enhancer elements. Particularly useful here are promoters that are functional in yeast.
Expression of a GPPS gene is determined by the promoter controlling the gene. In order for a gene to be expressed, a promoter must be present within 1,000 nucleotides upstream of the GPPS gene. A gene is generally cloned under the control of a desired promoter. The promoter regulates the amount of GPPS enzyme expressed in the cell and also the timing of expression, or expression in response to external factors such as sugar source.
Any promoter now known or later discovered can be utilized to drive the expression of the GPPS genes described herein. See e.g. http://parts.igem.org/Yeast for a listing of various yeast promoters. Exemplary promoters listed in Table 3 below drive strong expression, constant gene expression, medium or weak gene expression, or inducible gene expression. Inducible or repressible gene expression is dependent on the presence or absence of a certain molecule. For example, the GAL1, GAL7, and GAL10 promoters are activated by the presence of the sugar galactose and repressed by the presence of the sugar glucose. The HO promoter is active and drives gene expression only in the presence of the alpha factor peptide. The HXT1 promoter is activated by the presence of glucose while the ADH2 promoter is repressed by the presence of glucose.
In various embodiments, the nucleic acid is in a yeast expression cassette. Any yeast expression cassette capable of expressing GPPS in a yeast cell can be utilized. In some embodiments, the expression cassette consists of a nucleic acid encoding a GPPS with a promoter. Additional regulatory elements can also be present in the expression cassette, including restriction enzyme cleavage sites, antibiotic resistance genes, integration sites, auxotrophic selection markers, origins of replication, and degrons.
The expression cassette can be present in a vector that, when transformed into a host cell, either integrates into chromosomal DNA or remains episomal in the host cell. Such vectors are well-known in the art. See e.g. http://parts.igem.org/Yeast for a listing of various yeast vectors.
A nonlimiting example of a yeast vector is a yeast episomal plasmid (YEp) that contains the pBluescript II SK(+) phagemid backbone, an auxotrophic selectable marker, yeast and bacterial origins of replication and multiple cloning sites enabling gene cloning under a suitable promoter (see Table 3). Other exemplary vectors include pRS series plasmids.
The present invention is also directed to genetically engineered host cells that comprise the above-described nucleic acids. Such cells may be, e.g., any species of filamentous fungus, including but not limited to any species of Aspergillus, which have been genetically altered to produce precursor molecules, intermediate molecules, or cannabinoid molecules. Host cells may also be any species of bacteria, including but not limited to Escherichia, Corynebacterium, Caulobacter, Pseudomonas, Streptomyces, Bacillus, or Lactobacillus.
In some embodiments, the genetically engineered host cell is a yeast cell, which may comprise any of the above-described expression cassettes, and capable of expressing a GPPS comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or conservative amino acid substitutions to any one of the thirty-four sequences of SEQ ID NOs: 47-92.
Any yeast cell capable of being genetically engineered can be utilized in these embodiments. Nonlimiting examples of such yeast cells include species of Saccharomyces, Candida, Pichia, Schizosaccharomyces, Scheffersomyces, Blakeslea, Rhodotorula, or Yarrowia. These cells can achieve gene expression controlled by inducible promoter systems; natural or induced mutagenesis, recombination, and/or shuffling of genes, pathways, and whole cells performed sequentially or in cycles; overexpression and/or deletion of single or multiple genes and reducing or eliminating parasitic side pathways that reduce precursor concentration.
The host cells of the recombinant organism are engineered to produce any or all precursor molecules necessary for the biosynthesis of cannabinoids, including but not limited to olivetolic acid (OA), olivetol (OL), FPP and GPP, hexanoic acid and hexanoyl-CoA, malonic acid and malonyl-CoA, dimethylallylpyrophosphate (DMAPP) and isopentenylpyrophosphate (IPP) as disclosed in U.S. Pat. No. 10,435,727.
Construction of Saccharomyces cerevisiae strains expressing bacterial or archacal GPPS enzymes to produce GPP, NPP, FPP, and/or GGPP for cannabinoid and/or terpene production, such as CBGA or geraniol, is carried out via expression of a GPPS gene which encodes for an enzyme with GPPS activity such as the archacal (rkGPPS) and bacterial (bkGPPS) genes and proteins listed in Table 1. The GPPS gene can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the GPPS gene may be inserted into the recombinant host genome. Integration may be achieved by a single or double cross-over insertion event of a plasmid, or by nuclease based genome editing methods, as are known in the art e.g. CRISPR, TALEN and ZFR. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing. Sec, e.g., Green and Sambrook (2012)
In some embodiments, the recombinant cell further comprises a second recombinant nucleic acid that encodes a second enzyme in a terpenoid biosynthetic pathway. In some of these embodiments, the yeast cell is capable of expressing the second enzyme.
The second enzyme in these embodiments can encode any enzyme in the terpenoid biosynthetic pathway. In some embodiments, the second enzyme catalyzes synthesis of a compound that immediately precedes or is immediately after a product of the GPPS in the terpenoid biosynthetic pathway.
The recombinant cell can further comprise a third, fourth, etc. recombinant nucleic acid in the terpenoid biosynthetic pathway so that the cell can process a compound through at least three, four, five, etc. steps in the terpenoid biosynthetic pathway.
In some of these embodiments, the terpenoid biosynthetic pathway is not a cannabinoid biosynthetic pathway. In these embodiments, the recombinant cell can co-express genes for downstream terpenoid synthesis (reviewed in Davis and Croteau, 2000) such as cyclases, thiolases, desaturases, hydroxylases, hydrolases, oxidoreductases, and P450s, to produce monoterpenoids including but not limited to: 3-carene, ascaridole, bornane, borneol, camphene, camphor, camphorquinone, carvacrol, carveol, carvone, carvonic acid, chrysanthemic acid, chrysanthenone, citral, citronellal, citronellol, cuminaldehyde, p-cymene, cymenes, epomediol, cucalyptol, fenchol, fenchone, geranic acid, geraniol, geranyl acetate, geranyl pyrophosphate, grandisol, grapefruit mercaptan, halomon, hinokitiol, hydroxycitronellal, 8-hydroxygeraniol, incarvillateine, (s)-ipsdienol, jasmolone, lavandulol, lavandulyl acetate, levoverbenone, limonene, linalool, linalyl acetate, lineatin, p-menthane-3,8-diol, menthofuran, menthol, menthone, menthoxypropanediol, menthyl acetate, 2-methylisoborneol, myrcene, myrcenol, nerol, nerolic acid, ocimene, 8-oxogeranial, paramenthane hydroperoxide, perilla ketone, perillaldehyde, perillartine, perillene, phellandrene, picrocrocin, pinene, alpha-pinene, beta-pinene, piperitone, pulegone, rhodinol, rose oxide, sabinene, safranal, sobrerol, terpinen-4-ol, terpinene, terpineol, thujaplicin, thujene, thujone, thymol, thymoquinone, umbellulone, verbenol, verbenone, and wine lactone.
In other embodiments, the recombinant cell can also co-express genes for downstream terpenoid synthesis to produce sesquiterpenoids including but not limited to: abscisic acid, amorpha-4,11-diene, aristolochene, artemether, artemotil, artesunate, bergamotene, bisabolene, bisabolol, bisacurone, botrydial, cadalene, cadinene, alpha-cadinol, delta-cadinol, capnellene, capsidiol, carotol, caryophyllene, cedrene, cedrol, copaene, cubebene, cubebol, curdione, curzerene, curzerenone, dictyophorine, drimane, elemene, farnesene, farnesol, farnesyl pyrophosphate, germacrene, germacrone, guaiazulene, guaiene, guaiol, gyrinal, hernandulcin, humulene, indometacin farnesil, ionone, isocomene, juvabione, khusimol, koningic acid, ledol, longifolene, matricin, mutisianthol, nardosinone, nerolidol, nootkatone, norpatchoulenol, onchidal, patchoulol, periplanone b, petasin, phaseic acid, polygodial, rishitin, α-santalol, β-santalol, santonic acid, selinene, spathulenol, thujopsene, tripfordine, triptofordin c-2, valencene, velleral, verrucarin a, vetivazulene, a-vetivone, zingiberene.
In further embodiments, the recombinant cell can also co-express genes for downstream terpenoid synthesis to produce diterpenoids including but not limited to: abietane, abictic acid, ailanthone, andrographolide, aphidicolin, beta-arancosene, bipinnatin j, cafestol, cannabigerolic acid, carnosic acid, carnosol, cembratrienol, cembrene a, clerodane diterpene, crotogoudin, 10-deacetylbaccatin, elisabethatriene, crinacine, ferruginol, fichtelite, forskolin, galanolactone, geranylgeraniol, geranylgeranyl pyrophosphate, gibberellin, ginkgolide, grayanotoxin, guanacastepene a, incensole, ingenol mebutate, isocupressic acid, isophytol, isopimaric acid, isotuberculosinol, kahweol, labdane, lagochilin, laurenene, levopimaric acid, menatetrenone, mezerein, momilactone b, neotripterifordin, 18-norabietane, paxilline, phorbol, phorbol 12,13-dibutyrate, phorbol esters, phyllocladane, phytane, phytanic acid, phytol, phytomenadione, pimaric acid, pristane, pristanic acid, prostratin, pseudopterosin a, retinol, salvinorin, saudin, sclarene, sclareol, shortolide a, simonellite, stemarene, stemodene, steviol, taxadiene, taxagifine, taxamairin, taxodone, tenuifolin, 12-o-tetradecanoylphorbol-13-acetate, tigilanol tiglate, totarol, tricholomalide, tripchlorolide, tripdiolide, triptolide, triptolidenol.
In further embodiments, the recombinant cell can also co-express genes for downstream terpenoid modification to produce terpenoid derivatives including but not limited to: cholesterol, steroid hormones and analogs, heme, antioxidants such as carotenoids and quinones.
In specific embodiments, the recombinant cell is capable of producing nerol, geraniol, pinene, limonene, linalool, neral, citral, myrcene, ocimene, zingiberene, patchoulol, bisabolene, humulene, camphor, sabinene, geranylgeraniol, phytol, geranyllinalool, retinol, or any combination thereof.
The production of specific terpenes in recombinant cells can be enhanced by the use of specific recombinant GPPSs that preferentially produces geranyl pyrophosphate (GPP) or farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). For example, to enhance production of a monoterpene, the use of a GPPS that preferentially produces geranyl pyrophosphate (GPP) over farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) is beneficial. Similarly, to enhance production of a sesquiterpene, the use of a GPPS that preferentially produces FPP over GPP or GGPP is beneficial. Also, to enhance production of a diterpene, the use of a GPPS that preferentially produces GGPP over GPP or FPP is beneficial.
In various embodiments, the terpenoid biosynthetic pathway engineered in the recombinant host cell is a cannabinoid biosynthetic pathway. In these embodiments, the cell is capable of producing cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), cannabichromenic acid (CBCA), cannabinerolic acid (CBNA), cannabigerolic acid (CBGA), cannabinerovarinic acid (CBNVA), cannabigerophorolic acid (CBGPA), cannabigerovarinic acid (CBGVA), cannabigerogerovarinic acid (CBGGVA), tetrahydrocannabinolic acid (THCA), cannabinerovarinic acid (CBNVA), sesquicannabigerol (CBF), cannabigerogerol (CBGG), sesqui-cannabigerolic acid (CBFA), cannabigerogerolic acid (CBGGA), sesquicannabigerolic acid (CBFA), sesquicannabidiolic acid (CBDFA), sesquiTHCA (THCFA), sesqui-cannabigerovarinic acid (CBFVA), sesquiCBCA (CBCFA), sesquiCBGPA (CBFPA) or any combination thereof.
To enhance production of a cannabinoid, the use of a GPPS that preferentially produces GPP over FPP is beneficial.
The present invention is also directed to a method of producing a terpene in a yeast. The method comprises incubating any of the recombinant yeast cells described above in a manner sufficient to produce the terpene.
In some embodiments, a mixture of different archacal GPPS (rkGPPS) genes are expressed, a mixture of different bacterial GPPS (bkGPPS) genes are expressed, or a mixture of rkGPPS and bkGPPS are expressed in a modified strain. GPPS genes, such as those listed in Table 1, are synthesized using DNA synthesis techniques known in the art. The rkGPPS and bkGPPS genes can also be expressed in combination with known fungal GPPSes, such as Erg20 and the Erg20 mutants, and other fungal GPPSes (Genbank Accession Identification numbers: AFC92798.1, OBZ88092.1, AMM73096.1, EMS20556.1, CDR39302.1, ATB19148.1, AAY33922.1, ALK24263.1, ALK24264.1). Wild type ERG20 has the following corresponding GenBank
Accession Identification Number: CAA89462.1. Certain point mutations in ERG20 have been shown to change product specificity. Examples include: any combination of A99 to C, I, For W, and F96W and N127W as reported in Ignea (2014), mutation of A99 to any residue as reported in Rubat (2017) and mutation of K197 to any residue as reported in Fischer (2011) especially K197E and K197G. The optimized genes can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter and terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the optimized prenyltransferase genes are inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmids. Strains with the integrated genes can be screened by rescue of auxotrophy and genome sequencing.
In some embodiments, a monoterpene is produced. In some of these embodiments, a recombinant GPPS that preferentially produces GPP over FPP or GGPP is utilized. In other embodiments, a sesquiterpene is produced. In some of these embodiments, a recombinant GPPS that preferentially produces FPP over GPP or GGPP is utilized. In additional embodiments, a diterpene is produced. In some of these embodiments, a recombinant GPPS that preferentially produces GGPP over GPP and FPP is utilized.
Depending on the desired target molecule, it may be beneficial to selectively produce or increase GPP, FPP, or GGPP levels or modulate the ratio of GPP:FPP, GPP:GGPP, or FPP:GGPP to selectively obtain a desired end product (see
For the biosynthesis of phytocannabinoids such as CBG, CBD, CBC, and THC, the presence of farnesyl pyrophosphate (FPP) is undesirable as it may be combined with the prenyl acceptor molecule in place of GPP, yielding an undesirable sesquicannabinoid byproduct. To maximize production of cannabinoids such as THC and CBD, the concentration of GPP should be maximized and the concentration of FPP minimized. The pathway making both GPP and FPP in fungi is the mevalonate pathway, whose end product is ergosterol. In this pathway, GPP is the immediate precursor of FPP. However, GPP and FPP are synthesized by the same enzyme in yeast, Erg20, making it challenging to manipulate the Erg20 enzyme to produce predominantly GPP or predominantly FPP.
In yeast, some mutant alleles of the ERG20 gene use steric hindrance in the prenyl donor binding site of the enzymes to bias the synthase towards producing more GPP than FPP. The endogenous copy or copies of ERG20 can be replaced entirely by an engineered version of ERG20 to remove or greatly reduce the endogenous capacity to make FPP. While protein engineering approaches have been very successful in conferring specificity for GPP production over FPP, some of these mutations negatively affect the catalytic efficiency and catalytic rate of the enzyme (Ignea, 2013 and Rubat, 2017). Although not as catalytically efficient as the wild type enzyme, the engineered yeast enzyme can be used in combination with bacterial or archacal GPP synthases disclosed herein to increase the concentration of GPP while maintaining specificity (see
Conversely, FPP pools in an engineered host cell can be increased by certain other mutations of the endogenous Erg20. The engineered Erg20 fungal GPPS may be used in combination with a bacterial or archaeal enzyme that preferentially synthesizes FPP (
Pathways for GPP biosynthesis differ in other kingdoms. Bacteria use the methyl erythritol phosphate pathway, using entirely different biosynthetic enzymes and intermediates to make GPP. Archaca have a modified form of the mevalonate pathway (Vinokur, 2014). This presents the possibility that GPP synthase homologs derived from bacteria and archaca may have different GPP:FPP product ratios. Although they may also make FPP, some bacterial and archacal enzymes may have an advantage for GPP production, while others are more prone to generate FPP.
Thus, the set of recombinant heterologous enzymes disclosed offers a variety of options for constructing a modified host system biased either towards the production of FPP or the production of GPP. Choice of one set of enzymes should direct a cell towards making monoterpenoids or sesquiterpenoids.
To produce the desired terpene, each candidate polypeptide is introduced into a host cell genetically modified to contain all necessary components for cannabinoid and terpene biosynthesis using standard yeast cell transformation techniques (Green and Sambrook (2012). Cells are subjected to fermentation under conditions that activate the promoter controlling the candidate polypeptide (see, e.g., Table 3). The broth may be subsequently subjected to HPLC analysis (
DNA sequences encoding the GPPS are synthesized and cloned using techniques known in the art (Green and Sambrook (2012). Gene expression can be controlled by inducible or constitutive promoter systems (see Table 3) using the appropriate expression vectors. Genes are transformed into an organism using standard yeast or fungi transformation methods to generate modified host strains (i.e., the recombinant host organism). To produce cannabinoids, the modified strains which produce cannabinoid precursors express genes for (i) a bacterial GPP synthase, (ii) an archacal GPP synthase, or (iii) a mixture of archacal and bacterial GPP synthases to generate meroterpenoids such as CBGA, sesqui-CBGA, CBGGA, and mono-, sesqui-and di-terpenes. The modified strains from above can also co-express genes for downstream cannabinoid synthases, such as CBCA, THCA, and CBDA synthases, to produce additional cannabinoid compounds including but not limited to CBCA, CBCVA, CBC, THCA, THCVA, THCV, CBDA, CBDVA, CBD, CBGF, CBGFA, CBDF, CBDFA, THCF, THCFA, etc.
In some embodiments, recombinant heterologous GPPS genes are expressed in combination with a modified cannabinoid producing strain.
Construction of a modified Saccharomyces cerevisiae host is carried out by co-expressing cannabinoid synthases with (i) a rkGPPS enzyme, (ii) a bkGPPS enzyme, (iii) a mixture of either rkGPPS, bkGPPS, or both rkGPPS and bkGPPS enzymes, as shown in
During cannabinoid biosynthesis a polyprenyl pyrophosphate such as GPP, NPP, FPP, and GGPP acts as a prenyl donor and is combined with a prenyl acceptor to produce a cannabinoid. For example, combining GPP with olivetolic acid (OA) results in the formation of cannabigerolic acid (CBGA) (
When FPP is used in place of GPP during CBG biosynthesis, a prenylog is generated, published as sesquicannabigerol (CBF) (Pollastro, 2011). If the prenylog sesquicannabigerol (CBF) is the desired reaction product, in this case it would be desirable to increase intracellular levels of FPP. This could be accomplished by overexpression of bacterial and archacal GPP synthase enzymes (GPPSes) that preferentially make FPP.
When GGPP is used in place of GPP during CBGA and CBG biosynthesis, the prenylogs cannabigerogerol (CBGG) and cannabigerogerolic acid (CBGGA) are generated. If the prenylogs CBGG and CBGGA are the desired reaction products, in this case it would be desirable to increase intracellular levels of GGPP. This could be accomplished by overexpression of bacterial and archacal GPP synthase enzymes (GPPSes) that preferentially make GGPP.
CBGA is a precursor molecule of many downstream cannabinoids, e.g. CBDA, THCA, CBCA. If FPP is used in place of GPP in the biosynthesis of CBGA and the CBGA prenylogs sesquicannabigerol (CBF) or sesquicannabigerolic acid (CBFA) are generated (
The alkyl chain of the prenyl acceptor may also vary during cannabinoid biosynthesis. If divarinolic acid, also called divarinic acid or varinolic acid, which has an alkyl chain 2-carbons shorter than olivetolic acid (
Preferred embodiments are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.
Recombinant Saccharomyces cerevisiae were modified to express multiple GPPS genes, following the techniques described in Ignea (2014) and Rubat (2017).
Modification of host cells included expression of genes on self-replicating vectors and/or genetic insertion of recombinant genes by single or double cross-over insertion. Vectors used for modified host cell expression of GPPSes and biosynthetic pathways for terpenes and cannabinoids contained a yeast origin of replication, a promoter upstream of the recombinant gene or fusion-gene, and a poly-A terminator downstream of the recombinant genes or fusion-genes, allowing for expression of recombinant enzymes and fusion-enzymes (Table 1 and 2). In some cases, the vectors contained auxotrophic and drug-resistant markers for host cell selection, such as selectable cassettes for the amino acid, tryptophan, or antibiotic, geneticin. Recombinant genes were cloned into expression vectors using restriction digest and T4 ligation, by techniques known in the art.
The production of cannabinoids, sesquicannabinoids and terpenes by strains with various recombinant GPPSes is shown in
Construction of Saccharomyces cerevisiae strains expressing bacterial or archacal GPPS enzymes fused with N terminal cofolding peptides from Table 2. SEQ76-SEQ80 to produce GPP. NPP, FPP, and/or GGPP for cannabinoid and/or terpene production, including CBGA or geraniol, was carried out via expression of a fusion GPPS gene of any codon optimized nucleic acid sequence SEQ71-SEQ75 combined at the 5′ end of any nucleic acid sequence SEQ1-SEQ36 which encodes for an enzyme with GPPS activity such as the archacal (rkGPPS) and bacterial (bkGPPS) genes and proteins listed in Table 1. The fusion GPPS genes were cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid was confirmed by DNA sequencing. Alternatively, the fusion GPPS genes were inserted into the recombinant host genome. Integration was achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene were screened by rescue of auxotrophy and genome sequencing.
Cannabinoid-producing strains expressing the GPPSs of the present invention were grown in a feedstock as described in U.S. patent application Ser. No. 17/068,636, in a minimal-complete or rich culture media containing yeast nitrogen base, amino acids, vitamins, ammonium sulfate, and a carbon source, such as glucose or molasses. The feedstock was consumed by the modified host to convert the feedstock into (i) biomass, (ii) GPP, NPP, FPP, cannabinoids and/or terpenes, and (iii) biomass and GPP, NPP, FPP, cannabinoids and/or terpenes. Strains expressing the recombinant GPPS genes were grown on feedstock for 12 to 160 hours at 25-37° C. for isolation of products.
To identify fermentation-derived terpenes, cannabinoids, and sesquicannabinoids, (see
In view of the above, it will be seen that several objectives of the invention are achieved and other advantages attained.
As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
All references cited in this specification, including but not limited to patent publications and non-patent literature, are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.
As used herein, in particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. That the upper and lower limits of these smaller ranges can independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
The indefinite articles “a” and “an,” as used herein in the specification and in the embodiments, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the embodiments, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements can optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including clements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the embodiments, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the embodiments, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the embodiments, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the embodiments, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements can optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
This application claims the benefit of U.S. Provisional Application No. 63/141,486, filed Jan. 26, 2021, and incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/13857 | 1/26/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63141486 | Jan 2021 | US |
